1. Field of the Invention
The present invention relates to L-asparagine amidohydrolytic enzymes, more particularly, to polypeptides which originate from mammal, having L-asparaginase activity.
2. Description of the Prior Art
L-Asparaginase (EC 3.5.1.1) is an enzyme which catalyzes the hydrolytic reaction of L-asparagine into L-aspartic acid and ammonia. The studies on the antitumor activity of L-asparaginase started from the following reports: J. G. Kidd et al. described the inhibitory action of guinea pig sera on cells of lymphomas in xe2x80x9cThe Journal of Experimental Medicinexe2x80x9d, Vol.98, pp.565-582 (1953), and J. D. Broome et al. evidenced in xe2x80x9cNaturexe2x80x9d, Vol.191, pp.1,114-1,115 (1961), that the L-asparaginase activity of the guinea pig sera was responsible for the inhibitory action. It is now understood that the inhibitory action is caused by the lack of L-asparagine, an essential nutrient to proliferate and survive for some tumor cells which defect L-asparagine synthetase activity, such as acute lymphocytic leukemia, but not for normal cells. The hydrolysis of L-asparagine by L-asparaginase in patients with such tumor cells induces selective death of the tumor cells, resulting in the treatment of malignant tumors.
L-Asparaginase has been studied energetically for its actual use as an antitumor agent, and one derived from Escherichia coli is now in use as a therapeutic agent for leukemia and lymphoma. However, L-asparaginase from Escherichia coli is merely an external protein for human, and repetitive administration of conventional compositions with such L-asparaginase may cause serious side effects such as anaphylaxis shock, urticaria, edema, wheeze and dyspnea. These compositions are inevitably restricted with respect to administration dose and frequency. Therefore, some proposals to reduce or even diminish such side effects have been given.
As a first proposal, Japanese Patent Kokai No.119,082/79 discloses a chemically modified L-asparaginase from Escherichia coli, in which at least 65% amino acids are blocked with 2-O- substituted polyethylene glycol-4,6-dichloro-S-triazine. As a second proposal, human L-asparaginases are disclosed in Japanese Patent Kokai Nos.320,684/92 and 19,018/80, where the L-asparaginases are respectively obtained from cultures of human cell lines and human urine. While the first proposal has an advantage of that the L-asparaginase from Escherichia coil is easily obtainable on an industrial scale, it has a disadvantage of that the modifying reaction is difficult to control and the side effects couldn""t be eliminated completely. While the second proposal has an advantage of that unlike L-asparaginase from Escherichia coli, the L-asparaginases from human may not substantially induce antibodies even when administered to patients, it has a disadvantage of that it is not easy to obtain the L-asparaginases in a desired amount by the processes disclosed in Japanese Patent Kokai Nos.320,684/92 and 19,018/80.
Recently, recombinant DNA technology has advanced remarkably. If a DNA which encodes a desired polypeptide is once isolated, it is relatively easy to obtain a transformant which produces the polypeptide by constructing a recombinant DNA, comprising the DNA and a self-replicable vector, followed by introducing the recombinant DNA into a host, such as a microorganism, animal- or plant-cell. The polypeptide is obtainable in a desired amount from the culture of the transformant. However, no DNA which encodes mammalian L-asparaginase was isolated, and no mammalian L-asparaginase was produced by recombinant DNA techniques.
Therefore, it has been in great demand to isolate DNAs which encode active L-asparaginases originating from mammal and establish processes to prepare the L-asparaginases on a large-scale by applying the recombinant DNA techniques to the isolated DNAs.
In view of foregoing, the first object of the present invention is to provide a polypeptide which originates from mamma, having L-asparaginase activity.
The second object of the present invention is to provide a DNA which encodes the polypeptide.
The third object of the present invention is to provide a recombinant DNA which containing a DNA which encodes the polypeptide and a self-replicable vector.
The fourth object of the present invention is to provide a transformant obtainable by introducing a DNA which encodes the polypeptide into a host.
The fifth object of the present invention is to provide a process to prepare the polypeptide by using the transformant.
The sixth object of the present invention is to provide an agent for susceptive diseases, containing the polypeptide as an effective ingredient.
The first object of the present invention is attained by polypeptides which originate from mammal, having L-asparaginase activity.
The second object of the present invention is attained by DNAs which encode the polypeptides.
The third object of the present invention is attained by recombinant DNAs containing DNA which encode the polypeptides and a self-replicable vector.
The fourth object of the present invention is attained by transformants obtainable by introducing the DNAs into appropriate hosts.
The fifth object of the present invention is attained by a process to prepare the polypeptides which comprises culturing the transformants and collecting the produced polypeptides from the resultant cultures.
The sixth object of the present invention is attained by agents for susceptive diseases, containing the polypeptides as effective ingredients.